Dosing patients with gene transfer vectors for the treatment of lung disease likely will require intraluminal delivery strategies. We hypothesize that vector delivery via this route will confront two major barriers prior to any potential vector interaction with a cell surface receptor: (1) the transported mucus layer; (2) the cell surface tethered mucin/glycocalyx layer. Surprisingly little is known about the relative efficiencies (defined as percent of delivered vectors reaching epithelial cell surfaces) of the two principal delivery modes (aerosolization; lavage) for topical airways vector delivery. Thus, prior to further human clinical studies, we propose first to quantitate the barriers to vector penetration to the epithelial cell surface afforded by mucus clearance after aerosol vs. lavage vector delivery. Because we speculate that clearance of topically delivered vector will be rapid, and hence delivery to epithelial cell surfaces inefficient, we propose strategies to increase the efficiency for both aerosol and lavage administration, and use these data to select an optimal delivery system for our mouse studies (see below). Next, we hypothesize that vectors that escape mucus clearance will confront a second barrier, the cell surface -glycocalyx. Thus, we propose to identify the components of the glycocalyx that contribute to the functional barrier to gene transfer and design strategies to abrogate these barriers in studies with well-differentiated and freshly excised human airway epithelial preparations. The concepts and strategies to abrogate the barrier function of the glycocalyx, particularly the contribution of the tethered mucins MUC1 and MUC4, will be extended to in vivo conditions using transgenic mice. Employing a defined target (GPI-CAR) for adenovirus mediated gene transfer in the apical membrane of airway epithelia in transgenic mice, we will systematically explore the role of the glycocalyx as a barrier to gene transfer in wild-type mice and mice deficient in MUC1 and MUC4.